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Prevalence of Rota Virus Detection by Reverse Transcriptase

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Prevalence of Rota Virus Detection by Reverse Transcriptase IOSR Journal of Pharmacy and Biological Sciences (IOSR-JPBS) e-ISSN:2278-3008, p-ISSN:2319-7676. Volume 12, Issue 1 Ver. III (Jan. - Feb.2017), PP 94-99 www.iosrjournals.org Prevalence of Rota Virus Detection by Reverse Transcriptase- Polymerase Chain Reaction in Stool from Children Fewer Than Five Age With Acute Diarrhea in Diyala Province in Iraq Ansam Dawod Salman Lecturer in Diyala University, Collage of science, Department of Biology Abstract: The present study was conducted for the period from 1/6/2016 to 20/1/2017 in Baquba city. The study aimed to detection of rotavirus in stool specimens of children fewer than five age and also explore the effects of certain demographic factors on the detection rates by revers transcriptase- polymerase chain reaction. The study included 49 patients with acute diarrhea, 32 were male and 17 were female. The age range was two months to 5 years. Demographic information on the patients regarding age, sex, residence, type of feeding and source of drinking water were collected from their parents. Stool specimens were collected from each patients and. Detection of rotavirus in stool specimens was done by conventional reverse transcriptase polymerase chain reaction (RT-PCR). The results of present study showed that the overall infection rate by rotavirus among patients with acute diarrhea by RT-PCR tests was 93.88%. The highest infection rate was recorded among those >10-≤15 months of age. None of the results showed significantly difference between female and male, PCR (88% vs 96.87%). Likewise, there was insignificantly difference between urban and rural residence, PCR (95.65% vs 92.30%). The results revealed insignificantly higher infection rate among patients (those below 2 years) feed mixing (91.66%) and bottled (100%) compared to that breast feeding (77.77%) by RT-PCR. The rotavirus infection rate was insignificantly higher among patients consuming municipal water for drinking (97.22%) compared to those consuming bottled water (84.61%) by the RT-PCR. The study concluded that rotavirus was detected in high rates among children less than 5 years old with acute diarrhea in Baquba city, particularly those less than 2 year old. Keywords: Rotavirus, RT-PCR, diarrhea. I. Introduction The RV belongs to the Reoviridae family, exhibits icosahedral symmetry; it is not enveloped and was first identified by electron microscopy by Bishop et al. (1993)(1). The viral particle consists of three layers of protein and the viral genome consists of 11 segments of double-stranded RNA (dsRNA), which encode six structural proteins, VP1-4, VP6 and VP7 and six non-structural proteins, NSP1-6 (2). The VP6 protein, located in the inner capsid of the virus contains the antigenic determinants, which allow their classification into seven serogroups of A to G, with group A being the most common agent of childhood diarrhea (3). At present, 5 rotavirus serotypes (G1, G2, G3, G4, G9) are the predominant circulating strains, accounting for approximately 95% of strains worldwide, although there is considerable geographic variability (4 and 5). Today, Rotaviruses are recognized as the single most significant cause of severe gastroenteritis, malnutrition and diarrhea in young children in both developed and developing countries worldwide (6). Although RV can infect older children and adults, diarrheal disease is primarily observed in children under 2 years of age (7). It accounts for 5% of all deaths in children younger than 5 years in developing countries. Mortality rates in developed countries on the other hand are very low and illness is usually self limiting (8 and 9). RVs are transmitted by fecal-oral spread, and possibly also by the respiratory route (10). It has an incubation period of 18-36 hours. Following ingestion, the virus attaches to and then replicates in the differentiated villous columnar epithelial cells (enterocytes) lining the upper small intestine. Death and desquamation of infected enterocytes impairs the normal digestive and absorptive processes, outcomes a watery diarrhea of 2-7 days duration, resulting in loss of fluids and electrolytes, which may lead to severe or fatal dehydration. Rotavirus induced diarrhea is thought to be caused by a combination of factors (11), which include a reduction in epithelial surface area, replacement of mature enterocytes by immature cells, an osmotic effect resulting from incomplete absorption of carbohydrates from the intestinal lumen with bacterial fermentation of these non-absorbed compounds, secretion of intestinal fluid and electrolytes through activation of the enteric nervous system (12 and 13), and the effect of the RV non-structural protein 4 (NSP4), which is thought to act as a viral enterotoxin (14). DOI: 10.9790/3008-1201039499 www.iosrjournals.org 94 | Page Prevalence of Rota virus detection by reverse transcriptase- polymerase chain reaction in stool .. Initially, diagnosis of rotavirus infection was performed using electron microscopy, by visualization and observation of the rotavirus wheel-like appearance (15). Nowadays, the laboratory diagnosis of rotavirus infection is usually performed by antigen detection, using latex agglutination techniques, which is affirmed to be a good tool for the simple and rapid detection of RV in stool specimens (16 and 17). Enzyme-linked immunosorbant assays which are the most preferably used due to their high sensitivity and specificity for detection of RV in different pathological specimens (18 and 19). More sensitive and newer methods are molecular techniques such as polyacrylamide gel electrophoresis (PAGE) and reverse transcription-polymerase chain reaction (RT-PCR), which are used to determine the RV-RNA migration patterns and virus genotyping, respectively (20). II. Materials and Methods Study groups: The present study was conducted in Baquba city for the period from 1/6/2016 to 20/1/2017. The group of study were included; 49 patients with acute diarrhea. The patients were attended Al-Batool Maternity and Children Teaching Hospital and the following Primary Health Care Centers in Baquba (Al-Takea, Al-Sarai and Al-Tahrer). 32 of patients were males and 17 were females. The age range was 2 months to 5 years. Special questionnaire was pre-constructed to collect information regarding age, sex, residence, type of feeding and source of drinking water. The information was collected through short personal interview with patient's parents. Collection of stool specimens: Forty nine stool specimens were collected from patients were diagnosed by the doctor with disposable clean containers with tightly fixed caps. The specimens were transferred to the laboratory of Al-Batool Maternity and Children Teaching Hospital in a cool box as soon as possible. The PCR technique was carried out at Virology Laboratory/ College of Veterinary Medicine- Diyala University. Molecular detection of rotavirus: RNA extraction: RT-PCR test: This test was performed using commercially available kit (Sacace Biotechnologies / Rotavirus 280 PCR kit). Test principle: The test principle of the kit rotavirus 280 was based on four major processes: isolation of rotavirus RNA from specimens, reverse transcription of the RNA, amplification of the cDNA and detection of the amplified products on agarose gel. Interpretation of results: Analysis of PCR results were based on the presence or absence of specific bands of amplified DNA in the agarose gel (2%). The length of specific amplified DNA fragments was: * The specific bands of the rotavirus in the gel were 280 basepairs. * The specific bands of the rotavirus cDNA(+) in the gel were 280 base pairs. * The nonspecific bands of the DNA-buffer in the gel were not 280 base pairs. * Target region: NSP4. Statistical analysis: Data analyses were computer aided. Statistical analysis was done using SPSS (Statistical Package of Social Science) version 18 computer software. Frequency distribution and percentage for selected variables were done first. The Chi-Square test was used for association between two variables, and the P value (Probability) estimated was considered statistically significant if it's less than 0.05. Spearman correlation between screening test and different parameters was done by evaluate r value (correlation coefficient) and P value (Probability), and an estimate was considered statistically significant if P value less than 0.05. (21). III. Results Results presented below are based on statistical analyses of data accumulate throughout the study period. Detection of Rota virus: Polymerase chain reaction: Table (1) demonstrated that RV-RNA was detected in 46 out of 49 (93.88%) of the stool specimens by RT-PCR technique, while only 3(6.12%) specimens were negative. DOI: 10.9790/3008-1201039499 www.iosrjournals.org 95 | Page Prevalence of Rota virus detection by reverse transcriptase- polymerase chain reaction in stool .. Table (1): Results of PCR technique for human stool specimens Results Frequency % Human Positive 46 93.88 n=49 Negative 3 6.12 Effects of certain factors on PCR results: Results in table (2) showed that the RV-RNA detection rate was (100%) in three age groups; namely, >10 months - ≤15 months, >15 months -≤20 months, and >20months. Whereas, the lowest detection rate was among those ≤5 months (87.5%). Nevertheless, the difference among the age groups was statistically insignificant (p= 0.833). Table (2): Distribution of PCR results according to age. Result of PCR Age (months) P Value No. positive (%) No. negative (%) Total ≤5 months 7(87.5) 1(12.5) 8 >5 months - ≤10 months 26(92.85) 2(7.14) 28 >10 months - ≤15 months 6(100) 0(0.00) 6 0.833 >15 months -≤20 months 4(100) 0(0.00) 4 >20 months 3(100) 0(0.00) 3 Total 46 3 49 The RV-RNA detection rate among female patients was lower (88.2%) as compared to that in male patients (96.8%). The difference between the two groups was statistically insignificant (p= 0.23), table (3). Table (3): Distribution of PCR results according to sex. Result of PCR Sex P Value No.
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